A precise enumeration of the number of sub-visible particles such as virus particles, virus-like particles, inorganic and polymeric beads and other nanoparticles and micro-particles from liquid samples is important in many processes. For example, modified virus vectors are commonly used in gene therapy applications. The number of active vectors per mL (the infectious titer of the virus sample) can be determined using standard infectivity assays. However, by using the currently available methods, it is not possible to precisely determine the total number of particles, including non-infectious particles, in the sample. The ratio of infectious over non-infectious particles provides invaluable information about the quality and efficacy of the final gene therapy product and the upstream development processes.
One major limitation of the currently available techniques, such as quantitative flow cytometry (QFCM), is that the nanoparticles of interest are not directly detected. Instead, the number of bound probes to a population of nanoparticles is quantitated. Since the number of probes that binds per nanoparticle varies, the precision of the conventional indirect techniques is typically low and dependent on the affinity between the specimen and probe. A technique where the nanoparticle of interest could be directly detected would overcome this limitation. Moreover, if the technique would be able to visualize the particles at sufficient resolution, individual particles could be identified based on their size and morphology and thus be directly enumerated. Even particles within clusters could be enumerated and estimated. This is not possible by using the currently available affinity methods or light scattering-based techniques.
The novel high-precision direct particle method of the present invention may be used to enumerate both inorganic and organic sub-visible particles, such as nanoparticles, from liquid samples. One important feature is that the specimens are applied on a well-defined and measurable footprint. Another important feature is that the specimens are more evenly distributed than what has been possible before and this reduces the need for sampling and it is now possible to conduct the analysis at a resolution where the individual particles can easily be identified. The sub-visible particles are directly detected without the need for signal probes and can be visualized in normal two-dimensional images. The particle quantification SEM (pqSEM) method of the present invention is preferably based on low-vacuum filtering, scanning electron microscopy (SEM) or other electron microscopy techniques and image analysis. The present invention can be used with or without internal standards, of which an example would be National Institute of Standards and Technology (NIST) characterized polystyrene beads.
The present invention provides a solution to the above described problems. More particularly, the method is for quantification of sub-visible particles. A filter membrane is provided that has a plurality of pores defined therethrough. The filter membrane is in operational engagement with a vacuum chamber. The pores are sealed with a sealant. A sample droplet, containing a liquid with sub-visible particles, is applied onto the filter membrane. The liquid dissolves the sealant in the pores located directly below the sample droplet. The liquid flows through the pores in which the sealant has been dissolved and the sub-visible particles remain on top of the filter membrane. The filter membrane, with the particles disposed thereon, is moved to an electron microscope and enumerated in images acquired in the microscope.
The method further comprises the step of pre-mounting a filter assembly, containing the filter membrane, onto a SEM support.
The method further comprises the step of placing a mounting tape on the SEM support.
The method further comprises the step of providing the SEM support, having an elongate channel defined therein, using an injector containing the sample droplet, and aligning the injector on top of an elongate channel prior to depositing the sample droplet on the filter membrane.
The method further comprises the step of connecting the SEM support to a vacuum chamber connected to a vacuum source and subjecting the filter membrane to a suction force via the elongate channel.
The method further comprises the step of depositing the sample droplet onto the filter membrane without the sample droplet touching any outside edge of the filter membrane.
The method further comprises the step of the liquid only dissolving the sealant in the pores disposed directly below the sample droplet while the adjacent pores on the side of the droplet remain sealed with the sealant because the liquid has not been in contact with the sealant disposed in those pores.
The method further comprises the step of the sub-visible particles forming a defined and measurable footprint on the filter membrane and acquiring a series of images of the particles from an outside periphery of the footprint to the center of the footprint.
The method further comprises the step of counting the particles in the electron microscopy images acquired at a resolution where the particles are clearly visible—either manually or automatically using image analysis methods.
The method further comprises the step of estimating the total area of the footprint on the filter membrane in microscopy images covering the whole footprint (either one low-magnification image covering the whole footprint or several higher magnification sub-images of the footprint stitched together).
The method further comprises the step of calculating the total number of particles in the sample from the area of the whole footprint and the number of particles per area unit derived from images at a resolution high enough to clearly see single particles.
The method further comprises the step of possibly compensating for uneven radial particle distribution of the particles in the footprint for which information is derived from acquiring a series of images from the periphery of the footprint through the center at a high enough magnification to clearly see individual particles.
The method further comprises the step of calculating the concentration of particles in the solution using the total particle estimate from the footprint; the applied volume and dilution of the liquid sample.
The method further comprises the step of using a diluent of the liquid to dissolve the sealant in the pores located directly below the sample droplet. The specimen should be in a liquid form and the diluent should be compatible with the diluent and have the property of effectively dissolving the sealant that is being used.
The method further comprises the step of using glycine as the sealant. Other sealants that could be used include, but are not limited to, trehalose/sucrose-based sealants.